pRetroX-Tet-On Advanced

pRetroX-Tet-On Advanced

pRetroX-Tet-On Advanced逆病毒载体基本信息：

pRetroX-Tet-On Advanced载体质粒图谱和多克隆位点信息：

pRetroX-Tet-On Advanced载体简介：

pRetroX-Tet-On Advanced载体描述

pRetroX-Tet-On Advanced is a Retro-X Q retroviral vector that expresses rtTA-Advanced, an improved version of the reverse tetracycline(Tet)-controlled transactivator protein rtTA (1–4). rtTA-Advanced is more sensitive to doxycycline (Dox) and yields lower background expression than the original rtTA. The rtTA-Advanced protein is a fusion of the Tet repressor (TetR) DNA-binding domain, and three minimal “F”-type transcriptional activation domains from the herpes simplex virus VP16 protein. The gene encoding rtTA-Advanced is completely synthetic, lacks cryptic splice sites, and utilizes human codon preferences for stable expression in mammalian cells. Expression of rtTA-Advanced is driven by the powerful, constitutively active CMV promoter.

As with all of our Retro-X Q vectors, pRetroX-Tet-On Advanced uses LTR self-inactivation to eliminate promoter interference. In LTR self-inactivation, the mechanism of viral integration is used to introduce a deletion into the 5’ LTR. As a result of this mutation, the 5’ LTR CMV/MSV promoter, which drives high expression of the complete viral genome in packaging cells, is inactive in stably transduced target cells. This allows the expression of rtTA-Advanced (from PCMV IE) to proceed unimpeded (5, 6). The vector also contains a neomycin resistance gene (Neor) that allows G418 selection of stably transduced cells. rtTA-Advanced and the Neor marker are coexpressed from a bicistronic transcript containing an internal ribosome entry site (IRES).This ensures that a high frequency of G418 resistant clones express the Tet-On Advanced transactivator.

pRetroX-Tet-On Advanced contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, a packaging signal (Ψ+), and a tRNA primer binding site. The vector also contains an SV40 origin of replication for plasmid propagation in mammalian cells that express SV40 T antigen. In addition, a pUC origin of replication and an E. coli Ampr gene allow for propagation and selection in bacteria.

The pRetroX-Tet-On Advanced vector is used in conjunction with Tet response vectors to create double stable Retro-X Tet-On Advanced cell lines. Such cell lines are essentially Doxycycline (Dox)-controlled gene expression systems in which the Tet response vector expresses a gene of interest under the control of a Tet-responsive element (TRE; e.g. PTight; 7, 8). In the presence of Dox, rtTA-Advanced binds to the TRE and activates expression of the gene of interest. In the absence of Dox, rtTA-Advanced is unable to bind to the TRE, and the system is inactive. Additional information on TRE-containing vectors and protocols describing how to construct a Retro-X Tet-On Advanced cell line can be found in the Retro-X Tet-On Advanced Inducible Gene Expression System User manual (PT3958-1).

Selection of Stable Transfectants

 Selectable marker: plasmid confers resistance to G418.

Propagation in E. coli

 Suitable host strains: DH5α, DH10B and other general purpose strains.

 Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

pRetroX-Tet-On Advanced载体序列：

ORIGIN

    1 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC

   61 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC

  121 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA

  181 TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC

  241 AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA

  301 TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC

  361 GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC

  421 AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC

  481 GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCAATAAAA GAGCCCACAA CCCCTCACTC

  541 GGCGCGCCAG TCTTCCGATA GACTGCGTCG CCCGGGTACC CGTATTCCCA ATAAAGCCTC

  601 TTGCTGTTTG CATCCGAATC GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT

  661 TGACTACCCA CGACGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATTTGG AGACCCCTGC

  721 CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCCA GCAACTTATC TGTGTCTGTC

  781 CGATTGTCTA GTGTCTATGT TTGATGTTAT GCGCCTGCGT CTGTACTAGT TAGCTAACTA

  841 GCTCTGTATC TGGCGGACCC GTGGTGGAAC TGACGAGTTC TGAACACCCG GCCGCAACCC

  901 TGGGAGACGT CCCAGGGACT TTGGGGGCCG TTTTTGTGGC CCGACCTGAG GAAGGGAGTC

  961 GATGTGGAAT CCGACCCCGT CAGGATATGT GGTTCTGGTA GGAGACGAGA ACCTAAAACA

 1021 GTTCCCGCCT CCGTCTGAAT TTTTGCTTTC GGTTTGGAAC CGAAGCCGCG CGTCTTGTCT

 1081 GCTGCAGCGC TGCAGCATCG TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC

 1141 TGAAAATTAG GGCCAGACTG TTACCACTCC CTTAAGTTTG ACCTTAGGTC ACTGGAAAGA

 1201 TGTCGAGCGG ATCGCTCACA ACCAGTCGGT AGATGTCAAG AAGAGACGTT GGGTTACCTT

 1261 CTGCTCTGCA GAATGGCCAA CCTTTAACGT CGGATGGCCG CGAGACGGCA CCTTTAACCG

 1321 AGACCTCATC ACCCAGGTTA AGATCAAGGT CTTTTCACCT GGCCCGCATG GACACCCAGA

 1381 CCAGGTCCCC TACATCGTGA CCTGGGAAGC CTTGGCTTTT GACCCCCCTC CCTGGGTCAA

 1441 GCCCTTTGTA CACCCTAAGC CTCCGCCTCC TCTTCCTCCA TCCGCCCCGT CTCTCCCCCT

 1501 TGAACCTCCT CGTTCGACCC CGCCTCGATC CTCCCTTTAT CCAGCCCTCA CTCCTTCTCT

 1561 AGGCGCCGGA ATTGAAGATC TGGGGGATCG ATCCTCTAGA GTCCGTTACA TAACTTACGG

 1621 TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT

 1681 ATGTTCCCAT AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC

 1741 GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG

 1801 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT

 1861 TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT

 1921 GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC

 1981 CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC

 2041 GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA

 2101 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG

 2161 ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCGG CCGGGAACGG TGCATTGGAA

 2221 CGGACCTGCA GGAATTGATC CGCGGCCGCA CCGGTAGGCC TCGTACGCTT AATTAACGGA

 2281 TCCACCATGT CTAGACTGGA CAAGAGCAAA GTCATAAACG GCGCTCTGGA ATTACTCAAT

 2341 GGAGTCGGTA TCGAAGGCCT GACGACAAGG AAACTCGCTC AAAAGCTGGG AGTTGAGCAG

 2401 CCTACCCTGT ACTGGCACGT GAAGAACAAG CGGGCCCTGC TCGATGCCCT GCCAATCGAG

 2461 ATGCTGGACA GGCATCATAC CCACTTCTGC CCCCTGGAAG GCGAGTCATG GCAAGACTTT

 2521 CTGCGGAACA ACGCCAAGTC ATTCCGCTGT GCTCTCCTCT CACATCGCGA CGGGGCTAAA

 2581 GTGCATCTCG GCACCCGCCC AACAGAGAAA CAGTACGAAA CCCTGGAAAA TCAGCTCGCG

 2641 TTCCTGTGTC AGCAAGGCTT CTCCCTGGAG AACGCACTGT ACGCTCTGTC CGCCGTGGGC

 2701 CACTTTACAC TGGGCTGCGT ATTGGAGGAA CAGGAGCATC AAGTAGCAAA AGAGGAAAGA

 2761 GAGACACCTA CCACCGATTC TATGCCCCCA CTTCTGAGAC AAGCAATTGA GCTGTTCGAC

 2821 CGGCAGGGAG CCGAACCTGC CTTCCTTTTC GGCCTGGAAC TAATCATATG TGGCCTGGAG

 2881 AAACAGCTAA AGTGCGAAAG CGGCGGGCCG GCCGACGCCC TTGACGATTT TGACTTAGAC

 2941 ATGCTCCCAG CCGATGCCCT TGACGACTTT GACCTTGATA TGCTGCCTGC TGACGCTCTT

 3001 GACGATTTTG ACCTTGACAT GCTCCCCGGG TAACTAGAAT TCCGCCCCCC CCCCCCCCCC

 3061 CCCTCTCCCT CCCCCCCCCC TAACGTTACT GGCCGAAGCC GCTTGGAATA AGGCCGGTGT

 3121 GCGTTTGTCT ATATGTTATT TTCCACCATA TTGCCGTCTT TTGGCAATGT GAGGGCCCGG

 3181 AAACCTGGCC CTGTCTTCTT GACGAGCATT CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA

 3241 ATGCAAGGTC TGTTGAATGT CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TTGAAGACAA

 3301 ACAACGTCTG TAGCGACCCT TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC

 3361 TGCGGCCAAA AGCCACGTGT ATAAGATACA CCTGCAAAGG CGGCACAACC CCAGTGCCAC

 3421 GTTGTGAGTT GGATAGTTGT GGAAAGAGTC AAATGGCTCT CCTCAAGCGT ATTCAACAAG

 3481 GGGCTGAAGG ATGCCCAGAA GGTACCCCAT TGTATGGGAT CTGATCTGGG GCCTCGGTGC

 3541 ACATGCTTTA CATGTGTTTA GTCGAGGTTA AAAAAACGTC TAGGCCCCCC GAACCACGGG

 3601 GACGTGGTTT TCCTTTGAAA AACACGATGA TAAGCTTGCC ACAACCATGG CTGAACAAGA

 3661 TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC

 3721 ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC

 3781 GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC

 3841 GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC

 3901 TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC

 3961 TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC

 4021 GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG

 4081 TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT

 4141 CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGCGCATG CCCGACGGCG AGGATCTCGT

 4201 CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG

 4261 ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC

 4321 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG

 4381 TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG

 4441 AGCGGGATCG GCTAGAGTCG ACCCGGACGG GACGCTCCTG CGCCTGATAC AGAACGAATT

 4501 GCTTGCAGGC ATCTCATGAG TGTGTCTTCC CGTTTTCCGC CTGAGGTCAC TGCGTGGATG

 4561 GAGCGCTGGC GCCTGCTGCG CGACGGCGAG CTGCTCACCA CCCACTCGAG GGCTGCAGCG

 4621 CTGCAGAGGC CGAGTGCAGA ACTGCTCCAA AGGGACCTCA AGGCTTTCCG AGGGACACTA

 4681 GGCTGACTCC ATCGAGCCAG TGTAGAGATA AGCTTATCGA TTAGTCCAAT TTGTTAAAGA

 4741 CAGGATATCA GTGGTCCAGG CTCTAGTTTT GACTCAACAA TATCACCAGC TGAAGCCTAT

 4801 AGAGTACGAG CCATAGATAA AATAAAAGAT TTTATTTAGT CTCCAGAAAA AGGGGGGAAT

 4861 GAAAGACCCC ACCTGTAGGT TTGGCAAGCT AGCTTAAGTA ACGCCATTTT GCAAGGCATG

 4921 GAAAAATACA TAACTGAGAA TAGAGAAGTT CAGATCAAGG TCAGGAACAG ATGGAACAGG

 4981 GTCGACCCTA GAGAACCATC AGATGTTTCC AGGGTGCCCC AAGGACCTGA AATGACCCTG

 5041 TGCCTTATTT GAACTAACCA ATCAGTTCGC TTCTCGCTTC TGTTCGCGCG CTTCTGCTCC

 5101 CCGAGCTCAA TAAAAGAGCC CACAACCCCT CACTCGGGGC GCCAGTCCTC CGATTGACTG

 5161 AGTCGCCCGG GTACCCGTGT ATCCAATAAA CCCTCTTGCA GTTGCATCCG ACTTGTGGTC

 5221 TCGCTGTTCC TTGGGAGGGT CTCCTCTGAG TGATTGACTA CCCGTCAGCG GGGGTCTTTC

 5281 ATTTGGGGGC TCGTCCGGGA TCGGGAGACC CCTGCCCAGG GACCACCGAC CCACCACCGG

 5341 GAGGTAAGCT GGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA

 5401 GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA

 5461 GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC ATGACCCAGT CACGTAGCGA

 5521 TAGCGGAGTG TAGATCCGGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG

 5581 CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG

 5641 AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC

 5701 AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA

 5761 TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCGGC

 5821 CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA

 5881 GCTTACTGGC TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG

 5941 TGTGAAATAC CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCTC TTCCGCTTCC

 6001 TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA

 6061 AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA

 6121 AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG

 6181 CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG

 6241 ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT

 6301 CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT

 6361 TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC

 6421 TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT

 6481 GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT

 6541 AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC

 6601 TACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA

 6661 AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT

 6721 TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT

 6781 ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA

 6841 TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA

 6901 AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC

 6961 TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT

 7021 ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC

 7081 TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT

 7141 GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA

 7201 AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTGCAGG CATCGTGGTG

 7261 TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT

 7321 ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC

 7381 AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT

 7441 ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC

 7501 TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAACACG GGATAATACC

 7561 GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA

 7621 CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC

 7681 TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA

 7741 AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT

 7801 TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA

 7861 TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT

 7921 GACGTCTAAG AAACCATTAT TATCATGACA TTAACCTATA AAAATAGGCG TATCACGAGG

 7981 CCCTTTCGTC TTCAAGAATT AGCTTGGCCA TTGCATACGT TGTATCCATA TCATAATATG

 8041 TACATTTATA TTGGCTCATG TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT

 8101 TATTAATAGT AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GT//

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